This invention is directed to the field of molecular biology in general, and, more specifically, to a polypeptide associated with endometriosis, an isolated polynucleotide encoding the polypeptide, and methods of using these molecules.
Endometriosis is a painful disorder that is characterized by the ectopic implantation of functioning endometrial tissue into the abdominal wall and the outer surface of various organs including, most commonly, the lower bowel, ovaries and fallopian tubes. P. Vigano et al. (1991) Fertility and Sterility 56:894. Currently, endometriosis-specific genes have not been identified and the events relating to the development of endometriosis are poorly understood. However, several reports suggest that retrograde menstruation linked with abnormal immune function may play a role in establishing ectopic endometrium lesions. T. Ishimaru and H. Masuzaki (1991) Am. J. Obstet. Gynecol. 165:210-214. Many attempts to isolate antigens from ectopic endometrium lesions have failed, due to the necrotic nature of the lesions.
Endometriosis also is recognized has having an autoimmune component. IgG and IgA auto-antibodies that react with multiple endometrial antigens have been documented in patients with endometriosis. However, attempts to develop IgG or IgA-based assays for the diagnosis of endometriosis has fallen short of fruition. S. Fernandez-Shaw et al., (1996) Hum. Reprod. 11:180-1184. R. A. Wild et al. (1991) Obstetrics and Gynecology 77:927. Studies have shown that circulating IgG antibodies that bind multiple endometrial proteins can be detected in women with endometriosis to varying degrees. Thirty-five percent to 74% of patients have sera reactive with endometrial proteins. O. Odukoya et al. (1996) Acta Obstet. Gynecol. Scand. 75:927-931; J. G. Kim et al. (1995) Am. J. Reprod. Immunol. 34:80-87; O. A. Odukoya et al. (1995) Hum. Reprod. 10:1214-1219. It has also been shown that endometrial antibody titers in patients that respond well to danazol are significantly lower (7/18 (39%) treated patients had elevated titers) than those patients with untreated endometriosis or patients that responded poorly to treatment (17/23 (74%) untreated patients had elevated titers). A. El-Roeiy et al. (1988) Fertility and Sterility 50:864-871; H. J. Chihal et al. (1986) Fertility and Sterility 46:408-411. In addition, it has been recently reported that women with endometriosis have elevated levels of IL-4, a Th2 mediating cytokine, and that treatment with danazol reduces the levels of IL-4 in women that respond well to treatment. C.-C. Hsu et al. (1997) Fertility and Sterility 67:1059-1064.
This invention provides an isolated cDNA molecule and an alternately spliced variant encoding autoantigens associated with endometriosis. The autoantigen is called Repro-EN-1.0. The alternately spliced variant is called IB1. Subjects diagnosed with endometriosis have been found to have antibodies that specifically bind to Repro-EN-1.0 polypeptide and/or a IB1 polypeptide. These antibodies represent a highly sensitive and specific diagnostic marker for endometriosis. Recombinant Repro-EN-1.0 protein and recombinant IB1 protein are useful to detect such antibodies in immunoassays.
In one aspect this invention provides a recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide epitope of at least 5 amino acids of Repro-EN-1.0 (SEQ ID NO:2), or IB1 (SEQ ID NO:4) wherein the epitope specifically binds to antibodies from subjects diagnosed with endometriosis. In one embodiment the nucleotide sequence is selected from the Repro-EN-1.0 sequence of SEQ ID NO: 1 or IB1 sequence of SEQ ID NO: 3. In another embodiment the nucleotide sequence is identical to nucleotides 176 to 2755 of SEQ ID NO:1 or nucleotides 176 to 2986 of SEQ ID NO:3. In another embodiment the polynucleotide further comprises an expression control sequence operatively linked to the nucleotide sequence.
In another aspect this invention provides a polynucleotide primer pair which amplifies a nucleotide sequence encoding a polypeptide epitope of at least 5 amino acids of Repro-EN-1.0, or IB1 wherein the epitope specifically binds to antibodies from subjects diagnosed with endometriosis. The pair comprises: 1) a 3xe2x80x2 primer of at least 7 nucleotides that specifically hybridizes to a 3xe2x80x2 end of the nucleotide sequence or downstream from the sequence, and 2) a 5xe2x80x2 primer of at least 7 nucleotides that specifically hybridizes to the 3xe2x80x2 end of the complement of the nucleotide sequence or downstream from the complement of the sequence.
In another aspect this invention provides a recombinant cell comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence encoding a polypeptide epitope of at least 5 amino acids of Repro-EN-1.0 (SEQ ID NO:2), or IB1 (SEQ ID NO:4), wherein the epitope specifically binds to antibodies from subjects diagnosed with endometriosis.
In another aspect this invention provides a method for detecting a target polynucleotide comprising a nucleotide sequence selected from Repro-EN-1.0 cDNA (SEQ ID NO:1) or its complement, or IB1 cDNA (SEQ ID NO:3) or its complement in a sample. The method comprises the steps of: (a) contacting the sample with a polynucleotide probe or primer comprising a sequence of at least 7 nucleotides that specifically hybridizes to the nucleotide sequence and (b) detecting whether the probe or primer has specifically hybridized to the target polynucleotide, whereby specific hybridization provides a detection of the target polynucleotide in the sample.
In another aspect this invention provides a purified, recombinant Repro-EN-1.0 polypeptide whose amino acid sequence is identical to that of SEQ ID NO:2, or an allelic variant of SEQ ID NO:2, or an IB1 polypeptide whose amino acid sequence is identical to that of SEQ ID No:4, or an allelic variant of SEQ ID No:4.
In another aspect this invention provides a purified polypeptide comprising an epitope of at least 5 amino acids of Repro-EN-1.0 (SEQ ID NO:2), or an epitope of at least 5 amino acids of IB1 (SEQ ID NO: 4), wherein the epitope specifically binds to antibodies from subjects diagnosed with endometriosis.
In another aspect this invention provides a composition consisting essentially of an antibody that specifically binds to Repro-EN-1.0 polypeptide (SEQ ID NO:2), or IB1 polypeptide (SEQ ID NO:4).
In another aspect this invention provides a method for detecting a Repro-EN-1.0 polypeptide or IB1 polypeptide in a sample. The method comprises the steps of (a) contacting the sample with a ligand that specifically binds to the Repro-EN-1.0 polypeptide or IB1 polypeptide and (b) detecting specific binding between the ligand and Repro-EN-1.0 polypeptide or IB1 polypeptide. Specific binding provides a detection of Repro-EN-1.0 polypeptide or IB1 polypeptide in the sample. In one embodiment, the ligand is an antibody.
In another aspect this invention provides a method diagnosing endometriosis in a subject. The method comprises the steps of: (a) detecting a test amount of an antibody that specifically binds to Repro-EN-1.0 polypeptide or IB1 polypeptide in a sample from the subject; and (b) comparing the test amount with a normal range of the antibody in a control sample from a subject who does not suffer from endometriosis. A test amount above the normal range provides a positive indication in the diagnosis of endometriosis. The sample can be a blood product, e.g., serum, peritoneal fluid, menstrual fluid, vaginal secretion or urine. In one embodiment the antibody is an IgE, IgG or IgG4 immunoglobulin. In another embodiment the step of detecting comprises capturing the antibody from the sample with an immobilized Repro-EN-1.0 or a peptide comprising an epitope of Repro-EN-1.0 or IB1 or a peptide comprising an epitope of IB1, and detecting captured antibody. The step of detecting captured antibody can comprise contacting the captured antibody with a detectable antibody that specifically binds immunoglobulins and detecting binding between the captured antibody and the detectable antibody. In another embodiment the step of detecting can comprise capturing the antibody from the sample with an immobilized anti-immunoglobulin antibody and detecting captured antibody. The step of detecting captured antibody can comprise contacting the captured antibody with Repro-EN-1.0 or a polypeptide, or IB1 or a peptide comprising an epitope of IB1, comprising an epitope of Repro-EN-1.0 and detecting binding between the captured antibody and the Repro-EN-1.0 or polypeptide or IB1 or polypeptide.
In another aspect this invention provides a method for use in following the progress of endometriosis in a subject. The method comprises the steps of: (a) detecting first and second amounts of an antibody that specifically bind Repro-EN-1.0 polypeptide, or IB1 polypeptide, in samples from the subject at a first and a second time, respectively; and (b) comparing the first and second amounts. An increase between the first and second amounts indicates progression of the endometriosis and a decrease between the first and second amounts indicates remission of the endometriosis.
In another aspect this invention provides an isolated MHC-peptide complex comprising at least a portion of an MHC Class I molecule or an MHC Class II molecule, wherein the portion comprises a binding site that specifically binds a peptide having an amino acid binding motif specific to the molecule, and wherein the portion engages in CD4-mediated or CD8-mediated binding to T cells, and a peptide of at least 8 amino acids in a sequence selected from the amino acid sequence of Repro-EN-1.0 (SEQ ID NO:2) or IB1 (SEQ ID NO:4), wherein the peptide comprises the amino acid binding motif and comprises an epitope that specifically binds to a T cell receptor; wherein the complex specifically binds a T cell having a T cell receptor that specifically binds to the epitope, and wherein specific binding induces anergy in the T cell.
In another aspect this invention provides a method for treating endometriosis in a subject comprising the step of inhibiting an immune response against Repro-EN-1.0 or IB1 in the subject. The method can comprise administering to the subject an immunosuppressant in an amount effective to inhibit the immune response, administering to the subject an isolated MHC-peptide complex of the invention in an amount effective to inhibit the immune response or administering to the subject an anti-idiotypic antibody that specifically binds to an antigen binding site of an antibody that specifically binds to Repro-EN-1.0 or IB1 in an amount effective to inhibit the immune response.
In another aspect this invention provides a screening method for determining whether a compound increases or decreases the expression of Repro-EN-1.0 in a cell comprising contacting the cell with the compound and determining whether the production of Repro-EN-1.0 mRNA or polypeptide, or IB1 mRNA or polypeptide, are increased or decreased.
In another aspect this invention provides a method of detecting a chromosomal translocation of a Repro-EN-1.0 gene or IB1 gene comprising the steps of: a) hybridizing a labeled polynucleotide probe that specifically hybridizes with the Repro-EN-1.0 nucleotide sequence of SEQ ID NO:1 or its complement, or IB1 nucleotide sequence of SEQ ID NO:3 or its complement, to a chromosome spread from a cell sample to determine the pattern of hybridization and b) determining whether the pattern of hybridization differs from a normal pattern. A difference in the pattern provides detection of a translocation.
In another aspect this invention provides a method of detecting polymorphic forms of Repro-EN-1.0 or IB1 comprising the steps of: a) determining the identity of a nucleotide or amino acid at a selected position within the sequence of a test Repro-EN-1.0 gene or polypeptide, or IB1 gene or polypeptide; b) determining the identity of the nucleotide or amino acid at the corresponding position of native Repro-EN-1.0 (SEQ ID NO:1 or 2) gene or polypeptide, or IB1 (SEQ No:3 or 4) gene or polypeptide; and c) comparing the identity from the test gene or polynucleotide with the identity of the native gene or polypeptide, whereby a difference in identity indicates that the test polynucleotide is a polymorphic form of Repro-EN-1.0 or IB1.